Mulberry leaf antihypertensive active peptide as well as preparation method and application thereof

Abstract

The invention discloses a mulberry leaf antihypertensive active peptide as well as a preparation method and application thereof. According to the method, mulberry leaves are used as raw materials to prepare mulberry leaf albumin, enzymolysis parameters of enzymolysis time, pH, enzyme-substrate ratio and substrate concentration are optimized through a single factor experiment and an orthogonal experiment, and optimal enzymolysis conditions for preparing the ACE inhibitory peptide crude product are provided; the high-activity ACE inhibitory peptide is identified through further purification, polypeptide sequence determination and virtual screening, the ACE inhibitory peptide has extremely high solubility and good ACE inhibitory activity, and the IC50 values of the two peptides VPSCFDLTGK and RLPDFHGL with the highest activity are 8.23 [mu] mol / L (8.765 [mu] g / mL) and 23.01 [mu] mol / L (23.58 [mu] g / mL) respectively. The active peptide can be used as an angiotensin converting enzyme inhibitory peptide, and achieves the purpose of reducing blood pressure by inhibiting ACE activity.

Description

TECHNICAL FIELD

[0001] The application relates to the technical field of active peptides, in particular to a mulberry leaf blood pressure-lowering active peptide and a preparation method and application thereof. BACKGROUND

[0002] Primary hypertension is the most common type of hypertension, accounting for about 90%-95%. The pathophysiological factors of hypertension include the activation of the sympathetic nervous system, the renin-angiotensin-aldosterone system (RAAS) and inflammatory mediators, among which the renin-angiotensin-aldosterone system plays an important role in regulating blood pressure and fluid balance and is considered a therapeutic target for hypertension in clinical practice. At present, hypertension can be controlled by drugs, and these drugs are mostly synthetic angiotensin-converting enzyme (ACE) inhibitors, receptor blockers, diuretics, calcium channel blockers and peripheral adrenergic inhibitors. However, they have many side effects. Food proteins contain a large amount of bioactive peptides, many of which have been proven to have blood pressure-lowering ability. In previous studies, many peptides with ACE inhibitory properties have been discovered, isolated and identified. Compared with antihypertensive drugs, ACE-inhibitory peptides from food sources are generally safer. As a functional ingredient, food-derived ACE-inhibitory peptides have great potential for the treatment and prevention of hypertension. At present, blood pressure-lowering peptides are mainly derived from animal milk, livestock and poultry, fish, and plant grains, nuts and oilseeds. Plant protein peptides are widely available and have high activity, and are a hot research topic at present. Currently, plant-derived blood pressure-lowering peptides mainly include soybean peptides, rice bran peptides and nut peptides, and there is less research on leaf protein blood pressure-lowering peptides.

[0003] Mulberry as an important economic value of the plant, has a long history of cultivation in China, wide planting area, rich resources, raw materials easily. The traditional industry of mulberry leaves are mainly used as silkworm feed, low utilization, easy to cause a large amount of waste. Mulberry leaves contain alkaloids, flavonoids, polysaccharides, polyphenols, rich in protein and other substances, has a high nutritional value, in the traditional Chinese medicine treatment for the treatment of hypertension, hypoglycemic, hypolipidemic, improve cardiovascular disease, treatment of fever and liver protection, etc. The protein content of mulberry leaves is about 19%-27% of dry weight, is the leaf protein content of higher plants, and the content of mulberry leaf albumin is as high as 50%. In order to realize the maximum utilization of resources, there are many aspects of the activity of mulberry leaves, such as Chinese patent application CN107119097A with neutral protease alone or alkaline protease and neutral protease complex enzyme, and DEAESepharose Fast Flow anion column chromatography, Sephadex G-15 gel column chromatography and RP-HPLC for separation and purification preparation, get mulberry immune active peptide; Chinese patent application CN118830622A mulberry grinding after sterilization, inoculation of mixed spore suspension of coronaviruses for fermentation, then through ultrasonic extraction, vacuum concentration to obtain the concentrate, eluted after freeze drying to obtain the polyphenol extract with hypoglycemic function; Chinese patent application CN115998795A with volume fraction 80% of ethanol solution prepared specific external antioxidant activity of mulberry flavonoids; The current patent related to mulberry mainly focuses on the extraction of its phenolic, alkaloids and other active substances and the application research of antioxidant and hypoglycemic, there is little in-depth study on the antihypertensive activity of mulberry leaf albumin. SUMMARY

[0004] In order to overcome the problems existing in the prior art, one of the purposes of the present application is to provide a preparation method of mulberry antihypertensive active peptide.

[0005] The second purpose of the present application is to provide a mulberry antihypertensive active peptide.

[0006] The third purpose of the present application is to provide the application of the above-mentioned mulberry antihypertensive active peptide.

[0007] In order to achieve the above purpose, the technical scheme adopted by the present application is:

[0008] A preparation method of mulberry antihypertensive active peptide crude product, comprising the following steps:

[0009] Step 1: fresh mulberry leaves are washed, dried, crushed and sieved to obtain mulberry powder;

[0010] Step 2: After extracting the albumin of mulberry leaves, the albumin of mulberry leaves is dissolved in water to prepare a solution with a mass concentration of 0.5% to 5%, denatured at 95 to 100℃ for 5±0.5 min, and then cooled to add protease for enzymolysis, and after the enzymolysis, the enzyme activity is inactivated at 95 to 100℃ for 10±1 min to obtain an enzymolysis solution;

[0011] Step 3: After the enzymolysis solution is cooled, centrifugation is performed, and the supernatant is freeze-dried to obtain a crude product of the blood pressure-lowering active peptide of mulberry leaves.

[0012] Further, the method for extracting the albumin of mulberry leaves in Step 2 is water extraction-acid precipitation or water extraction-salt precipitation.

[0013] Further, the amount of water used in Step 2 is calculated based on a mass concentration of 1% of the albumin of mulberry leaves.

[0014] Further, the denaturation condition in Step 2 is denaturation at 95℃ for 5 min.

[0015] Further, the enzyme inactivation condition in Step 2 is enzyme inactivation at 95℃ for 10 min.

[0016] Further, the centrifugation condition in Step 3 is 5000 to 10000 g for 15 to 20 min.

[0017] Further, the protease is neutral protease or alkaline protease.

[0018] Further, the enzymolysis condition is neutral protease at 30 to 60℃ and pH 6 to 8, or alkaline protease at 30 to 50℃ and pH 8 to 12, and the enzymolysis time is 0.5 to 8 h, and E / S is 2000 to 10000 U / g.

[0019] Further, the enzymolysis condition is alkaline protease at 30 to 50℃ and pH 8 to 12, the enzymolysis time is 0.5 to 6 h, the enzyme-substrate ratio is 1% to 3.5%, and the substrate concentration is 0.5% to 3%.

[0020] Further, the enzymolysis condition is alkaline protease at 35 to 45℃ and pH 8 to 11, the enzymolysis time is 1 to 4 h, the enzyme-substrate ratio is 2% to 3.5%, and the substrate concentration is 0.5% to 2%.

[0021] Further, the enzymolysis condition is alkaline protease at 40±2℃ and pH 10±0.2, the enzymolysis time is 2±0.2 h, the enzyme-substrate ratio is 2.5±0.2%, and the substrate concentration is 1±0.1%.

[0022] A crude product of a blood pressure-lowering active peptide of mulberry leaves is obtained by the above preparation method.

[0023] A preparation method of a mulberry leaf blood pressure lowering active peptide, comprising all steps of the mulberry leaf blood pressure lowering active peptide enzymatic hydrolysate and the following steps:

[0024] Step 4: The mulberry leaf blood pressure lowering active peptide crude product is dissolved with water, filtered with a 0.45 μm filter membrane, and the filtrate is ultrafiltered to obtain a Mw<1 kDa component;

[0025] Step 5: The Mw<1 kDa component is separated and purified by Sephadex G-15 gel column chromatography, the separated components are collected, and UPLC-MS / MS is used for further identification and analysis, so as to obtain the mulberry leaf blood pressure lowering active peptide.

[0026] Further, the solution concentration of the Sephadex G-15 gel column chromatography in step 5 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 3-6 min / tube.

[0027] Further, the solution concentration of the Sephadex G-15 gel column chromatography in step 5 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 3.5 min / tube.

[0028] A mulberry leaf blood pressure lowering active peptide, which has an amino acid sequence as shown in any one of the following sequences: DGFALDLGRN, RLPDFHGL, APGGFRE, VPSCFDLTGK, APGFEGR, FPNPGYYDGR, FDRFGP, EFFELFKF, RFDFDPL, YGDFDFGGH, FRGGLR, FDRGSF, HPSPGSAHRF, FDPNLGGK, PPVHGRL, FDPVGLLPK, SFWDGK, EPFPLLPK, TGGWDFR, PDSFRPK, EPPFLDPK, KPGMFGR, GPGSNPGLK, DFPVGLLPK, TGGGPGFR.

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

[0030] Further, the amino acid sequence is as shown in any one of the following sequences: RLPDFHGL, VPSCFDLTGK.

[0031] The mulberry leaf blood pressure lowering active peptide crude product or the mulberry leaf blood pressure lowering active peptide is used for preparing an ACE inhibitor, a health care product for helping to maintain a healthy blood pressure level, and / or a blood pressure lowering drug.

[0032] Further, in the application, the ACE inhibitor, the health care product with the function of helping to maintain the healthy level of blood pressure and / or the antihypertensive drug is prepared by using the antihypertensive active peptide of mulberry leaves as an active ingredient, and food or pharmaceutically acceptable carriers or adjuvants can be added.

[0033] An ACE inhibitor, comprising the antihypertensive active peptide of mulberry leaves as an active ingredient.

[0034] Further, the ACE inhibitor further comprises pharmaceutically acceptable carriers or adjuvants.

[0035] A health care product with the function of helping to maintain the healthy level of blood pressure, comprising the antihypertensive active peptide of mulberry leaves as an active ingredient.

[0036] Further, the health care product further comprises food acceptable carriers or adjuvants.

[0037] An antihypertensive drug, comprising the antihypertensive active peptide of mulberry leaves as an active ingredient.

[0038] Further, the antihypertensive drug further comprises pharmaceutically acceptable carriers or adjuvants.

[0039] The application first determines the optimal enzymolysis condition through single factor test and orthogonal test, then carries out enzymolysis under the optimal enzymolysis condition, and then carries out separation and purification by using 10 kDa, 3 kDa and 1 kDa ultrafiltration membranes and a Sephadex G-15 gel column, respectively, so that the enzymolysis condition is optimized, the purification process is mild and simple, the ACE inhibitory activity of the mulberry leaf peptide is better maintained, and an optimal enzymolysis condition for preparing the mulberry leaf ACE inhibitory peptide is provided.

[0040] The mulberry leaf ACE inhibitory peptide prepared in the application has extremely strong solubility and good ACE inhibitory activity, can be used as a natural antihypertensive agent or health care product, and is well applied to the food and drug industry.

[0041] Specifically, the application uses mulberry leaves as raw materials to prepare mulberry leaf albumin by water extraction and acid precipitation or water extraction and salt precipitation, parameters such as enzymolysis time, pH, enzyme-substrate ratio and substrate concentration are optimized through single-factor experiments and orthogonal experiments, then the mulberry leaf albumin is separated and purified by ultrafiltration (1kDa-10kDa) and Sephadex G-15 gel column chromatography, 25 ACE inhibitory peptides with potential blood pressure lowering activity are screened out by polypeptidomics and molecular docking technology, and finally six peptides, namely DGFALDLGRN, RLPDFHGL, APGGFRE, VPSCFDLTGK, APGFEGR and FPNPGYYDGR, are obtained through solid-phase synthesis and ACE inhibitory activity determination, and the inhibition rates of the six peptides on ACE are 76.51%, 93.59%, 77.86%, 94.17%, 84.02% and 89.86% respectively at a concentration of 2mg / mL, and the IC 50 values of the two peptides VPSCFDLTGK and RLPDFHGL with the highest activity are 8.23μmol / L (i.e. 8.765μg / mL) and 23.01μmol / L (i.e. 23.58μg / mL) respectively. The above active peptides can be used as angiotensin converting enzyme (ACE) inhibitory peptides, and by inhibiting the activity of ACE, the purpose of lowering blood pressure can be achieved.

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

[0043] 1) The application optimizes the mulberry leaf enzymolysis conditions and provides an optimal enzymolysis condition for preparing ACE inhibitory peptide crude product.

[0044] 2) The application purifies and prepares mulberry leaf ACE inhibitory peptides, greatly enriches the research range of effective components of mulberry leaves, and strengthens the application value.

[0045] 3) The ACE inhibitory peptides prepared by the application have strong solubility and good ACE inhibitory activity.

[0046] 4) The application identifies 6 high-activity ACE inhibitory peptides through polypeptide sequence determination and virtual screening.

[0047] 5) The application provides a simple and feasible preparation method of mulberry leaf ACE inhibitory peptides.

[0048] 6) The preparation process of the application does not involve any organic solvent, the operation condition is mild, the ACE inhibitory activity of the mulberry leaf peptides is maximally retained, and the safety of the product is ensured. BRIEF DESCRIPTION OF DRAWINGS

[0049] Figure 1Figure of the ACE inhibitory activity of the hydrolysate under different conditions of enzyme hydrolysis time (A), pH (B), enzyme-substrate ratio (C) and substrate concentration (D); different letters represent significant differences in the ACE inhibitory activity of the hydrolysate under different conditions of enzyme hydrolysis, p<0.05;

[0050] Figure 2 Figure of the ACE inhibitory activity of the crude product of the mulberry leaf protein HP and different ultrafiltration fractions thereof;

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

[0052] Figure 4 Figure of the ACE inhibitory activity of the S1 and S2 components obtained by dextran gel separation and purification;

[0053] Figure 5 Figure of the ACE inhibitory activity of the positive control Captopril and the six polypeptides at a concentration of 2 mg / mL;

[0054] Figure 6 Figure of the IC value determination results of the polypeptides VPSCFDLTGK (A) and RLPDFHGL (B). 50 Figure of the IC value determination results of the polypeptides VPSCFDLTGK (A) and RLPDFHGL (B). DETAILED DESCRIPTION

[0055] In order to better understand the present application, the following examples are used to further describe the present application, but it should be noted that the examples do not constitute a limitation on the scope of protection of the present application.

[0056] Example 1

[0057] An enzymatic preparation of mulberry leaf ACE inhibitory activity peptides and process optimization (alkaline protease hydrolysis) includes the following steps:

[0058] (1) Enzymatic preparation of crude mulberry leaf ACE inhibitory activity peptides and process optimization: dissolve the mulberry leaf albumin in water to prepare a solution with a mass concentration of 1% (w / v), denature the protein at 95°C for 5 min, adjust the pH after cooling, add alkaline protease (source leaf biological), and hydrolyze at 40°C for a certain period of time. After hydrolysis, 95°C is used to inactivate the enzyme for 10 min. Cool to room temperature, centrifuge at 8000g for 15 min, take the supernatant, dialyze and desalt with a 100 Da dialysis bag, freeze-dry, and obtain the crude mulberry leaf ACE inhibitory activity peptides;

[0059] In the enzymatic preparation, the parameters of enzyme hydrolysis time, pH, enzyme-substrate ratio and substrate concentration are optimized by single factor experiment and orthogonal experiment, as follows:

[0060] 1) Single factor experiment

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

[0062] Effect of reaction system pH on ACE inhibition rate of enzymatic hydrolysis products: When substrate concentration was 1% (w / v), enzyme-substrate ratio was 2.5% (w / w), and enzymatic hydrolysis time was 2h, ACE inhibition rate at pH values ​​of 8, 9, 10, 11, and 12 was significantly higher (see results in Table 2). Figure 1 (B) The ACE inhibition rate of the hydrolysis products under different hydrolysis pH conditions first increased and then decreased with the increase of the hydrolysis pH. The ACE inhibition rate reached a maximum of 56.14% at a hydrolysis pH of 10. The hydrolysis pH of 10 was selected as the optimal hydrolysis pH for subsequent orthogonal experiments.

[0063] Effect of enzyme-substrate ratio on ACE inhibition rate of enzymatic hydrolysis products: When the substrate concentration was controlled at 1% (w / v), the enzymatic hydrolysis time was 2h, and the pH was 10, the ACE inhibition rate was 2%, 2.5%, 3%, and 3.5% respectively (see the results). Figure 1 (C) The ACE inhibition rate of the enzymatic hydrolysis products under different enzyme-substrate ratios initially increased and then decreased with increasing enzyme-substrate ratios, reaching a maximum of 55.34% at an enzyme-substrate ratio of 2.5%. Therefore, a 2.5% enzyme-substrate ratio was selected as the optimal enzymatic hydrolysis time for subsequent orthogonal experiments.

[0064] Effect of substrate concentration on ACE inhibition rate of enzymatic hydrolysis products: When the enzyme-substrate ratio was controlled to be 2.5% (w / w), the enzymatic hydrolysis time was 2h, and the pH was 10, the ACE inhibition rate was 0.5%, 1%, 1.5%, and 2% when the substrate concentration (w / v) was 0.5%, 1%, 1.5%, and 2% (see the results). Figure 1 (D) The ACE inhibition rate of the enzymatic hydrolysis products under different substrate concentration conditions 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 test

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

[0067] According to the results of single factor experiment, a four-factor three-level orthogonal experiment was designed, with the independent variables A, B, C and D representing the enzyme hydrolysis time (h), pH value, enzyme-substrate ratio (%) and substrate concentration, respectively. The ACE inhibition rate was used as the index to obtain the ACE inhibition rate of each treatment group (see Table 2). According to the results of intuitive analysis in Table 3, by comparing the range R values of the four factors, the enzyme hydrolysis time was the most important factor affecting the ACE inhibition activity. The primary and secondary factors affecting the ACE inhibition rate were in the order of time (A) > pH (B) > enzyme-substrate ratio (C) > substrate concentration (D). According to the results of variance analysis (Table 4), the enzyme hydrolysis time (A) and pH value (B) had significant effects on the ACE inhibition rate of the crude mulberry leaf peptide HP, while the enzyme-substrate ratio (C) and substrate concentration (D) had no significant effects. Based on the above results, A1 (enzyme hydrolysis time of 2 h) and B3 (pH of 10) were determined as the optimal levels, and the enzyme-substrate ratio and substrate concentration had less effect on the ACE inhibition rate of the crude mulberry leaf peptide HP. Therefore, C2 (enzyme-substrate ratio of 2.5%) and D1 (substrate concentration of 1%) were selected. Thus, the optimal scheme was A1B3C2D1, i.e., enzyme hydrolysis time of 2 h, enzyme hydrolysis pH of 10, enzyme-substrate ratio of 2.5%, and substrate concentration of 1%. The verification experiment of the optimized enzyme hydrolysis conditions showed that the ACE inhibition activity of the mulberry leaf ACE inhibitory peptide prepared under the optimal enzyme hydrolysis conditions was 56.53%.

[0068] Table 1 L9(3 4 ) Orthogonal experiment factor level table

[0069]

[0070] Table 2 Orthogonal experiment scheme and results

[0071]

[0072]

[0073] Table 3 Orthogonal model intuitive analysis

[0074]

[0075] Table 4 Orthogonal model variance analysis

[0076]

[0077] (2) Isolation and purification of mulberry leaf ACE inhibitory peptides: The mulberry leaf ACE inhibitory peptide crude product obtained under the optimal enzymatic condition in (1) was dissolved in deionized water to prepare a 1 mg / mL solution, and filtered with a 0.45 μm filter. 10 mL of the filtrate was passed through 10 kDa, 3 kDa, and 1 kDa ultrafiltration membranes, respectively, and four fractions were collected, namely HP1 (Mw>10 kDa), HP2 (3 kDa Figure 2 ) It can be seen that ultrafiltration significantly improves the ACE inhibitory rate of the mulberry leaf peptide crude product HP, and the ACE inhibitory rate of HP4 is the highest, reaching 74.19%, which is 20.07% higher than that of HP.

[0078] The most active component HP4 was separated and purified by Sephadex G-15 gel column chromatography, the sample concentration was 6.8 mg / mL, the sample volume was 2 mL, purified water was used for elution, the elution flow rate was 0.5 mL / min, and 3.5 min / tube. The separated components S1, S2 Figure 3 ) were freeze-dried, and the ACE inhibitory activity was determined (results as shown in Figure 4 ), and the most active component was S2, which was 78.22%, i.e., a mulberry leaf ACE inhibitory peptide.

[0079] (3) Determination of polypeptide sequence: The polypeptide component 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, and then detected by UPLC-MS / MS. The collected data were processed by MM File Conversion file conversion software, converted into MGF format files, and 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 was a liquid chromatography-mass spectrometry system composed of an Easy-nLC 1200 ultra-high performance liquid chromatograph and an Orbitrap Eclipse DDA high-resolution mass spectrometer. The chromatographic column was an Acclaim PepMap RSLC C18 chromatographic column 2 pmol / L, 0.075 mm x 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. The peptides separated by UPLC were directly introduced into MS for online detection. From the sequenced peptides, those not modified by functional gene groups with peak area greater than 1 x 10 5 A total of 6341 polypeptide sequences were identified.

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

[0081] (4) Using molecular docking technology, 25 peptides with potential antihypertensive activity were screened from the 6341 polypeptides identified in (3), and the biological prediction activity, water solubility and binding affinity results 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 Biological prediction activity, water solubility and binding affinity

[0083]

[0084]

[0085] (5) Polypeptide synthesis: The antihypertensive peptides with the highest potential activity screened in (4) were synthesized by FMOC solid-phase synthesis method, and were desalted to a purity of ≥98%, and were synthesized by Shengong Bioengineering (Shanghai) Co., Ltd.

[0086] (6) ACE inhibition activity determination

[0087] 1) Preparation of reaction solution

[0088] A certain amount of sample was dissolved in sodium phosphate buffer, filtered, and prepared into a solution of the corresponding concentration. 15 μL of ACE solution was added to the sample tube and the blank tube, 30 μL of ACE inhibitor 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 HHL solution was added, and after reaction at 37°C for 1 h, 150 μL of 1.0 mol / L HCl solution was added to stop the reaction, to obtain a reaction solution, as shown in Table 6 below.

[0089] Table 6 Preparation of reaction solution

[0090]

[0091] 2) Chromatographic conditions Chromatographic column: ECOSIL C18 (260 mm x 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°C; 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 is rapidly decomposed by the catalysis of ACE to produce hippuric acid (Hip) and dipeptide (His-Leu, HL), and hippuric acid has a maximum absorption at 228 nm. When ACE inhibitor samples are added, the activity of ACE enzyme is inhibited, and the amount of hippuric acid generated is reduced, so the activity of ACE inhibitors can be evaluated by determining the amount of hippuric acid generated by high performance liquid chromatography. Inhibition rate of ACE activity. The calculation formula is: R = (A0-B) / A0 x 100%, where: R: inhibition rate of ACE inhibitor peptide sample on ACE (%); B: peak area of hippuric acid in the ACE inhibitor peptide group; A0: 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 IC 50 .

[0094] The results show that at a concentration of 2 mg / mL, the inhibition rates of the six peptides on ACE are 76.51%, 93.59%, 77.86%, 94.17%, 84.02%, and 89.86%, respectively, and the IC 50 values of the two peptides with the highest activity, VPSCFDLTGK and RLPDFHGL, are 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 the preferred embodiments of the present application, but the embodiments of the present application are not limited to the above-described embodiments, and any changes, modifications, substitutions, combinations, simplifications, etc. made without departing from the spirit and principles of the present application should be equivalent replacement manners and should be included in the protection scope of the present application.

Claims

1. A method for preparing a crude product of a mulberry leaf antihypertensive active peptide, characterized in that: The steps include: Step 1: Wash and dry fresh mulberry leaves, crush them and sieve them to obtain mulberry leaf powder; 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 at 95 to 100° C. for 5±0.5 minutes, add protease for enzymatic hydrolysis after cooling, and inactivate the enzyme at 95 to 100° C. for 10±1 minutes to obtain an enzymatic hydrolyzate; Step 3: After cooling the enzymatic hydrolysate, centrifuge it, take the supernatant and freeze-dry it to obtain the crude product of mulberry leaf antihypertensive active peptide.

2. The method for preparing the crude product of mulberry leaf antihypertensive active peptide according to claim 1, wherein: The method for extracting mulberry leaf albumin described in step 2 is water extraction and acid precipitation method or water extraction and salt precipitation method; The amount of water in step 2 is calculated based on the mass concentration of mulberry leaf albumin being 1%; The denaturation conditions described in step 2 are: denaturation at 95°C for 5 min; The enzyme inactivation conditions described in step 2 are: 95°C for 10 min; The centrifugal conditions described in step 3 are: 5000-10000 g, 15-20 min.

3. The method for preparing the crude product of the mulberry leaf antihypertensive active peptide according to claim 1 or 2, characterized in that: The enzymatic hydrolysis conditions are: alkaline protease, 40±2° C., pH 10±0.2, enzymatic hydrolysis time 2±0.2 h, enzyme-substrate ratio 2.5±0.2%, and substrate concentration 1±0.1%.

4. A crude product of mulberry leaf antihypertensive active peptidase, characterized by: The method is obtained by the preparation method described in any one of claims 1 to 3.

5. A method for preparing a mulberry leaf blood pressure-lowering active peptide, characterized in that: The method comprises all the steps of the preparation method according to any one of claims 1 to 3 and the following steps: Step 4: The crude product of mulberry leaf antihypertensive active peptide is dissolved in water and filtered through a 0.45 μm filter membrane. The filtrate is ultrafiltered to obtain a fraction with Mw < 1 kDa; Step 5: The fraction with Mw < 1 kDa is separated and purified by Sephadex G-15 gel column chromatography, the separated fractions are collected, and further identified and analyzed by UPLC-MS / MS to obtain the mulberry leaf antihypertensive active peptide; The solution concentration of the Sephadex G-15 gel column chromatography described in step 5 is 5-30 mg / mL, the sample volume is 1-5 mL, the elution is done with pure water, the elution flow rate is 0.3-0.8 mL / min, and the time is 3-6 min / tube.

6. A mulberry leaf antihypertensive active peptide, characterized by: The method is obtained by the preparation method described in claim 5.

7. A mulberry leaf blood pressure lowering active peptide, characterized in that: Its amino acid sequence is shown in any of the following sequences: DGFALDLGRN, RLPDFHGL, APGGFRE, VPSCFDLTGK, APGFEGR, FPNPGYYDGR, FDRFGP, EFFELFKF, RFDFDPL, YGDFDFGGH, FRGGLR, FDRGSF, HPSPGSAHRF, FDPNLGGK, PPVHGRL, FDPVGLLPK, SFWDGK, EPFPLLPK, TGGWDFR, PDSFRPK, EPPFLDPK, KPGMFGR, GPGSNPGLK, DFPVGLLPK, TGGGPGFR.

8. The mulberry leaf antihypertensive active peptide according to claim 7, characterized in that: Its amino acid sequence is shown in any of the following sequences: DGFALDLGRN, RLPDFHGL, APGGFRE, VPSCFDLTGK, APGFEGR, FPNPGYYDGR; preferably RLPDFHGL, VPSCFDLTGK.

9. Use of the crude product of the mulberry leaf antihypertensive active peptide according to claim 4 or the mulberry leaf antihypertensive active peptide according to any one of claims 6 to 8 in the preparation of ACE inhibitors, health products that help maintain healthy blood pressure levels, and / or antihypertensive drugs.

10. A product characterized by: The product is any one of the following products: Product 1: an ACE inhibitor comprising the mulberry leaf antihypertensive active peptide according to any one of claims 6 to 8 as an active ingredient; Product 2: A health product that helps maintain healthy blood pressure levels, comprising the mulberry leaf antihypertensive active peptide described in any one of claims 6 to 8 as an active ingredient; Product 3: A blood pressure lowering drug comprising the mulberry leaf blood pressure lowering active peptide described in any one of claims 6 to 8 as an active ingredient.

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