Method for preparing moringa oleifera seed polypeptide for lowering blood pressure and use thereof

Abstract

The application belongs to the technical field of plant source bioactive peptide development, and discloses a preparation method and application of a moringa oleifera seed blood pressure reducing polypeptide. A moringa oleifera seed polypeptide with angiotensin converting enzyme (ACE) inhibiting activity is separated and purified from moringa oleifera seed crude protein, and through structure identification, activity screening and solid phase synthesis, nine sequences of novel moringa oleifera seed ACE inhibiting peptides with blood pressure reducing effects are finally obtained, and the sequence of the polypeptide with the highest activity PLVRRAIY is reformed to improve its anti-trypsin enzymolysis performance. Molecular level and animal experiments show that the original peptide PLVRRAIY and the reformed peptide PPVQQAIY both have significant ACE inhibiting activity and blood pressure reducing effects, and the anti-trypsin degradation ability of the reformed peptide in the gastrointestinal environment is enhanced, and the stability is significantly improved, thereby providing a new resource for the development of natural blood pressure reducing polypeptide drugs.

Description

Technical Field

[0001] This invention belongs to the field of plant-derived bioactive peptide development technology, specifically relating to Moringa seed antihypertensive peptides, their preparation methods, and uses. Background Technology

[0002] Hypertension is a major risk factor for cardiovascular disease (CVD), causing irreversible damage to multiple organs during its development. It is a significant global health challenge affecting over 1.2 billion people worldwide. Blood pressure and cardiovascular function are regulated by multiple systems, among which the renin-angiotensin-aldosterone system (RAAS) is a crucial regulatory system. Angiotensin-converting enzyme (ACE) is a key regulatory target of the RAAS, responsible for catalyzing the conversion of inactive angiotensin I (Ang I) into the potent vasoconstrictor angiotensin II (Ang II), leading to elevated blood pressure. ACE inhibitors effectively block the conversion of Ang I to Ang II and are the main first-line drugs for treating hypertension, such as captopril, enalapril, and lisinopril. However, most existing drugs non-specifically inhibit the C and N domains of ACE, leading to common clinical side effects such as cough and angioedema. Recent studies have shown that specifically inhibiting the C domain of ACE is an effective strategy for treating hypertension and reducing these side effects. Currently, the evaluation system for plant-derived ACE inhibitory peptides is well-established, but reports on peptides that specifically inhibit the ACE C domain are still relatively few.

[0003] Moringa, a plant native to Africa Moringa oleiferaMoringa (L.) is a perennial tropical deciduous tree belonging to the Moringaceae family and the Moringa genus, widely cultivated in tropical and subtropical regions of Asia, Africa, and Central America. It was introduced to Yunnan Province, China in 1960, and is now also cultivated in Taiwan and Guangdong. Moringa seeds are rich in protein; defatted and fat-enriched moringa seeds contain 62.76% and 36.18% protein, respectively, and are rich in 17 amino acids, mainly including amino acids with the ability to donate protons and electrons: tyrosine, methionine, and cysteine; also containing relatively abundant hydrophobic amino acids: leucine, proline, phenylalanine, and valine; and abundant acidic amino acids: glutamic acid. Studies have confirmed that protease hydrolysis is a commonly used method to obtain ACE inhibitory peptides from raw proteins. Selective proteases that cleave amino acids at different sites selectively expose amino acids that contribute to ACE inhibitory activity. For example, the presence of aromatic amino acids or their location at the chain terminus, and the presence of phenylalanine, proline, and tyrosine at the C-terminus, will produce higher ACE inhibitory activity. Furthermore, the peptides obtained using the complex protease hydrolysis method exhibited significantly higher bioactivity than those obtained using the single protease hydrolysis method, and were also more readily available as small molecule peptides. Studies have found that hydrolyzing Moringa seed active proteins using the complex protease hydrolysis method can produce short peptides with good ACE inhibitory activity; however, only a few peptides have been identified, and research on peptides targeting the ACE C domain is lacking. Therefore, screening for naturally occurring, highly active, and specific ACE C domain inhibitory peptides from Moringa seeds can provide new resources for the discovery of active ingredients related to blood pressure health. Summary of the Invention

[0004] The main objective of this invention is to provide a moringa seed antihypertensive peptide, its preparation method, and its uses, aiming to provide a novel, natural, and less toxic plant-derived antihypertensive small molecule peptide.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] The preparation method of Moringa seed blood pressure lowering peptides includes the following steps:

[0007] (1) After defatting, the crude protein of Moringa seeds was extracted with 1M Tris-HCl solution at pH 8.0. The crude protein was then enzymatically hydrolyzed with a combination of alkaline protease and trypsin to produce total Moringa seed polypeptides that specifically inhibit the activity of angiotensin-converting enzyme.

[0008] (2) Polypeptides with a molecular weight of less than 1000 Da that inhibit angiotensin-converting enzyme activity were isolated from total polypeptides of Moringa seeds by ultrafiltration.

[0009] Moringa seed peptides isolated using the above method, exhibiting angiotensin-converting enzyme (ACE) inhibitory activity, underwent structural identification, activity screening, and solid-phase synthesis. This resulted in nine novel ACE-inhibiting peptides from moringa seeds with antihypertensive effects, including peptides containing at least one of the following sequences: VTRLTRP, LAATHGLYVY, FFKRLLRDSK, LSSSLLCTTKLP, IAHHHLAIAILF, PLVRRAIY, PGRQPAFQ, PVMLVHFP, and SMNCMTPF. All nine novel moringa seed peptides exhibited significant ACE inhibitory effects. PLVRRAIY showed the highest ACE C domain inhibitory activity (77.4%) and the ACE N domain inhibitory activity (36.1%), indicating that PLVRRAIY selectively inhibits the ACE C domain.

[0010] To further enhance the antihypertensive effect of the small molecule peptide PLVRRAIY and improve its stability in the gastrointestinal environment, peptides PIVNNAIY, PVVNNAIY, PPVNNAIY, and PPVQQAIY were obtained through modification. Compared to the original PLVRRAIY peptide, the modified peptides are less susceptible to trypsin degradation and retain high ACE inhibitory activity. In animal studies, PLVRRAIY and its modified peptide PPVQQAIY exhibited significant antihypertensive effects, providing new resources for the development of antihypertensive products.

[0011] Compared with the prior art, the present invention has the following beneficial effects:

[0012] 1) The total polypeptides produced by the combined enzymatic hydrolysis of crude protein from Moringa seeds by alkaline protease and trypsin have the highest ACE inhibitory activity. Further comparison of polypeptides with different molecular weights revealed that Moringa seed polypeptides with a molecular weight of less than 1 kD after enzymatic hydrolysis by alkaline protease / trypsin have better ACE inhibitory activity.

[0013] 2) Moringa seed peptides with ACE inhibitory activity and a molecular weight of less than 1 kDa were separated and purified by dextran gel chromatography and semi-preparative liquid chromatography. Further screening revealed specific ACE inhibitory peptides. After structural identification and activity screening, nine novel natural moringa seed ACE inhibitory peptides with antihypertensive potential were obtained. Among them, peptide PLVRRAIY showed the strongest in vitro specific inhibitory activity against the ACE C domain, with an inhibition rate of 77.4%.

[0014] 3) To improve the stability of the peptide PLVRRAIY, this invention modifies its sequence, designing four modified peptides that are less susceptible to degradation by gastrointestinal proteases and maintain high levels of ACE inhibitory activity. Molecular and animal experiments both demonstrate that the original peptide PLVRRAIY and the modified peptide PPVQQAIY exhibit significant ACE inhibitory activity and antihypertensive effects. Furthermore, the modified peptides show enhanced resistance to trypsin degradation in the gastrointestinal environment, resulting in significantly improved stability. Attached Figure Description

[0015] Figure 1 Comparison of ACE inhibitory activities of single protease and complex protease hydrolysates.

[0016] Figure 2 This is a gel filtration chromatogram of dextran fractions with a Da content less than 1000.

[0017] Figure 3 This is a chromatogram of the components separated by semi-preparative liquid chromatography.

[0018] Figure 4 The ACE inhibitory activity of each component was determined by semi-preparative liquid chromatography.

[0019] Figure 5 To evaluate the in vitro ACE C domain inhibitory activity of the synthesized candidate peptides.

[0020] Figure 6 To evaluate the in vitro ACE N domain inhibitory activity of the synthesized candidate peptides.

[0021] Figure 7 This is a secondary mass spectrum of PLVRRAIY (MO-18).

[0022] Figure 8 This is a secondary mass spectrum of PPVQQAIY (MO-186).

[0023] Figure 9 This is a graph showing the changes in blood pressure in an acute animal experiment with PLVRRAIY (MO-18).

[0024] Figure 10 This is a graph showing the changes in blood pressure in an acute animal experiment with PPVQQAIY (MO-186). Detailed Implementation

[0025] The main experimental materials and reagents used in the embodiments of this invention are: Moringa seeds, collected in 2023 from the China-Africa Joint Center in Kenya; Alcalase, from R&D Systems. 2.4 U / g; Trypsin, Sigma-Aldrich, USA, 1000-2000 BAEE units / mg solid; Flavorzyme, R&D Systems. 500 U / g; α-chymotrypsin, Sigma-Aldrich, USA. 40 U / mg; pepsin, > 3000 U / mg; angiotensin-converting enzyme (ACE-1UN), Sigma-Aldrich, USA; hip-his-leucine (HHL), Sigma-Aldrich, USA.

[0026] The main instruments used in the embodiments of this invention are: high-speed refrigerated centrifuge (Shanghai Lishen Scientific Instruments Co., Ltd.); electric thermostatic water bath (Shanghai Jinghong Experimental Equipment Co., Ltd.); target protein rapid separation system (GE, Sweden); binary high-pressure semi-preparative liquid chromatograph (Wuhan Ruihe Chromatography Technology Co., Ltd.); and vacuum freeze dryer (Ningbo Xinzhi Biotechnology Co., Ltd.).

[0027] Example 1: Preparation of antihypertensive small molecule peptides from Moringa seeds and evaluation of their in vitro ACE inhibitory activity

[0028] In this embodiment, moringa seed crude protein was hydrolyzed using a single protease method and a complex protease method. The in vitro ACE inhibitory activity of the hydrolysate obtained at different time points was compared. It was found that the moringa seed small molecule peptides obtained by hydrolyzing moringa seed crude protein using the complex protease method reached the optimal in vitro ACE inhibitory activity in a shorter time.

[0029] Specific methods:

[0030] (1) Moringa seed pretreatment

[0031] Sun-dried moringa seeds, after removing the outer and inner seed coats, yield white, spherical kernels, which are then pulverized and passed through a 100-mesh sieve. The sieved powder is mixed with petroleum ether at a ratio of 1:8 (w / v) and defatted using ultrasound (100 W, 25℃) for 3 hours, with the petroleum ether being replaced 2-3 times during this process. Afterward, the mixture is allowed to stand and filtered, the petroleum ether is discarded, and a blocky powder is obtained. The petroleum ether is then completely evaporated, and the powder is sieved again to obtain defatted moringa seed powder, which is stored at -40℃ for later use.

[0032] (2) Extraction of crude protein from Moringa seeds

[0033] Mix defatted Moringa seed powder with 1 M Tris-HCl (pH 8.0) solution at a ratio of 1:12 (w / v). Let stand on ice for 3-4 hours, then centrifuge at 12,000 rpm for 30 minutes at 4°C. Repeat 2-3 times, collecting the supernatant as the crude protein solution. Dialyze the crude protein solution overnight using a 5 kDa dialysis bag to remove salts, changing the deionized water 2-3 times during the process. Collect the solution inside the dialysis bag, freeze-dry it, and store it at -40°C for later use.

[0034] (3) Enzymatic hydrolysis of crude protein from Moringa seeds

[0035] Three proteases—alcalase, trypsin, and flavorzyme—were selected for single-protease hydrolysis and dual-protease combination hydrolysis of crude protein from Moringa seeds. The optimal hydrolysis conditions are shown in Table 1.

[0036] The specific procedure for single protease hydrolysis is as follows: Weigh an appropriate amount of crude moringa seed protein and dissolve it in PBS to achieve a concentration of 5 mg / mL. After adjusting the pH and temperature to the optimal conditions for each protease, first incubate the crude moringa seed protein for 10 min, then immediately take samples, inactivate it at 100℃ for 10 min, and centrifuge at high speed to obtain the supernatant as a crude protein control. Then, add the protease and incubate for 0.1 h, 0.5 h, 1 h, 3 h, 5 h, 7 h, 12 h, and 24 h, respectively, and take samples after each incubation. Inactivate the protease at 100℃ for 10 min, centrifuge at high speed to obtain the supernatant as the hydrolysis samples at different time points, aliquot them, and store them at -20℃ for later use.

[0037] The specific procedures for the dual-protease combined hydrolysis are as follows: This experiment uses two different enzyme combinations: alkaline protease / trypsin and alkaline protease / flavor protease. An appropriate amount of crude moringa seed protein was weighed, dissolved in PBS, and its concentration was adjusted to 5 mg / mL. After adjusting the pH and temperature to the optimal conditions for alkaline protease, the crude moringa seed protein was incubated for 10 min and a sample was taken. The protein was then inactivated at 100℃ for 10 min, and the supernatant was collected by high-speed centrifugation as a crude protein control. Alkaline protease was then added, and after incubation for 2 h, a sample was taken as a control sample before the dual-protease hydrolysis. The alkaline protease hydrolysate was inactivated at 100℃ and cooled to room temperature. The hydrolysate was adjusted to the optimal pH and temperature for trypsin or flavor protease, respectively. Trypsin or flavor protease was added, and the samples were incubated for 0.1 h, 0.5 h, 1 h, 3 h, 5 h, 7 h, 12 h, and 24 h, respectively. Samples were taken and inactivated, and the supernatant was collected by high-speed centrifugation as hydrolysis samples at different time points. These samples were aliquoted and stored at -20℃ for later use.

[0038] Table 1 Optimal enzymatic hydrolysis conditions for different proteases

[0039]

[0040] (4) Comparison of the effects of different protease hydrolysates of Moringa seeds on ACE inhibitory activity

[0041] The in vitro ACE inhibitory activity of the products from Moringa seed protease hydrolysis at different time points was determined. 100 μL of 5 mM hippuryl-histyl-leucine (HHL) was mixed with 50 μL of hydrolysate, and a buffer solution containing 0.1 M boric acid and 0.3 M NaCl (pH 8.3) was added to bring the total volume to 300 μL. A control group without hydrolysate was set up, and captopril was used as a positive control. The mixture was incubated at 37°C for 20 min; then 30 μL of rabbit lung-derived ACE enzyme solution was added, and the reaction was incubated at 37°C for 1 h. Finally, 250 μL of 1.5 M HCl was added to terminate the reaction. Finally, add 1.5 mL of ethyl acetate and vortex the mixture for 2 min to extract hippuric acid. Centrifuge at 12,000 rpm, transfer 1 mL of the ethyl acetate layer to a new centrifuge tube, evaporate and concentrate, then resuspend in 200 μL of the initial mobile phase. Filter through a 0.22 μm PES membrane and detect by UPLC.

[0042] The UPLC detection method is as follows: CNW Athena C18 liquid chromatography column (4.6 × 150 mm, 3 μm, 120 Å); flow rate 0.3 mL / min; detection wavelength 228 nm; injection volume 20 μL; mobile phase 0.1% TFA water (A)-acetonitrile (B); elution gradient 0-13 min, 25-80% B, 13-15 min, 80-25% B.

[0043] The formula for calculating the ACE inhibition rate is as follows:

[0044]

[0045] In the formula: R: ACE inhibition rate of the ACE inhibitor (%)

[0046] A: Peak area of ​​the product in the blank control group

[0047] B: Peak area of ​​the product in the sample group.

[0048] Experimental results: Comparison of the ACE inhibitory activities of crude protein from Moringa seeds hydrolyzed by single and complex proteases as follows: Figure 1As shown, the ACE inhibition activity of the crude protein from Moringa seeds extracted with 1 M Tris-HCl (pH 8.0) was 70% in the sample hydrolyzed with alkaline protease / trypsin for 0.1 h, 64% in the sample hydrolyzed with alkaline protease for 0.1 h, 5.3% in the sample hydrolyzed with trypsin for 0.1 h, and -43% in the sample hydrolyzed with flavor protease for 0.1 h. Compared with the inhibition activity of samples hydrolyzed with a single protease, the ACE inhibition activity of the alkaline protease / trypsin combination hydrolysis was better than that of samples hydrolyzed with a single protease, and the time to reach the optimal inhibition activity was shortened.

[0049] Example 2: Isolation and purification of a novel Moringa seed ACE inhibitory peptide

[0050] In this embodiment, membrane separation technology (ultrafiltration), Sephadex G-50 gel filtration chromatography and semi-preparative liquid chromatography were used to further separate and purify the Moringa seed polypeptide sample extracted with 1 M Tris-HCl (pH 8.0) and digested with alkaline protease / trypsin combination for 0.1 h. The polypeptide component with the highest inhibitory activity was obtained by evaluating the in vitro ACE inhibitory activity.

[0051] (1) Preparation of Moringa seed polypeptide molecular weight fractionation based on ultrafiltration method

[0052] Ultrafiltration tubes with molecular weight cutoffs of 1, 3, 10, and 30 kDa were selected for ultrafiltration separation of highly active Moringa seed protein hydrolysate. The specific procedure is as follows: First, the ultrafiltration tubes were washed multiple times with deionized water, and the ultrafiltration membrane was cleaned by centrifugation. 5-10 mL of the hydrolysate was transferred to a 50 mL 30 kDa ultrafiltration tube and centrifuged at 4℃ and 5000 rpm for 30 min. During centrifugation, the ultrafiltration membrane was gently agitated continuously with a pipette to reduce sample adsorption or prevent membrane clogging. Centrifugation was repeated until the volume of the retentate in the inner tube no longer decreased, and the retentate in the inner tube was collected as a peptide solution > 30 kDa. The entire peptide solution in the outer tube was transferred to a 10 kDa ultrafiltration tube, centrifuged again, and the above steps were repeated. The liquid in the inner tube was then the 10 kDa-30 kDa fraction. The solution from the outer tube was transferred to an ultrafiltration tube with a 3 kDa cutoff, and this process was repeated until peptide solutions of 3-10 kDa, 1-3 kDa, and < 1 kDa were collected. The peptide solutions of different molecular weights obtained by ultrafiltration were lyophilized to obtain lyophilized peptide powders of different molecular weights. The in vitro ACE inhibitory activity of the lyophilized peptide powders of different molecular weights was determined by UPLC.

[0053] (2) Isolation and purification of Moringa seed peptides based on gel filtration chromatography

[0054] The ultrafiltration cutoff fraction with a high ACE inhibition rate (< 1 kDa) was separated and purified using a self-assembled Sephadex G-50 chromatography column on an AKTA pure 25 target protein rapid separation system.

[0055] Sephadex G-50 chromatography column packing steps:

[0056] Dextran gel pretreatment: At room temperature, soak 4 g of dextran gel powder in distilled water for at least 24 hours, stirring constantly to ensure gel swelling until it is fully swollen and its volume no longer changes. Before swelling, add distilled water and stir, then allow it to settle naturally. If there are many floating gel fragments in the upper layer after sedimentation, rinse repeatedly to remove them and prevent blockage of the gel column during chromatography, which would affect the flow rate. After swelling, wash repeatedly with distilled water 3-5 times to remove residual trace organic reagents and alkaline substances.

[0057] Column packing: Add the swollen gel to distilled water (distilled water: gel = 1:3), stir gently, and then degas using sonication. Place the prepared gel packing material into the column (Jiangsu Hanbang Technology LCC glass chromatography column, 10.0 × 400mm) in one go. Be careful to keep the column wet during packing and avoid air bubbles or discontinuities in the column. The packing should be uniform.

[0058] Equilibration: Before loading the sample, equilibrate the chromatography column with at least 3-5 column volumes of deionized water until the recorder baseline becomes stable.

[0059] The lyophilized sample was prepared into a 10 mg / mL solution using deionized water and filtered through a 0.22 μm PES (polyethersulfone) aqueous filter membrane. After baseline equilibration, the sample was loaded at a volume of 0.5 mL and a flow rate of 0.5 mL / min. Elution was performed isocratically with deionized water at a detection wavelength of 280 nm. The AKTA system monitored and plotted the UV absorption signal over time in real time. Samples were collected using an automated fraction collector, programmed to collect by volume (1 mL per tube), with one tube collected every 2 minutes, automatically switching to the next tube. After collection, the samples from each collection tube corresponding to each peak were mixed according to the UV absorption spectrum, then lyophilized to obtain three fractions, F1-F3. Figure 2 Among them, the F3 component showed better ACE inhibitory activity, with a retention time range of 28.67 min - 48.36 min and a peak area of ​​528.6 mAu*min.

[0060] (3) Separation and purification of Moringa seed polypeptides based on semi-preparative liquid chromatography

[0061] 1) Sample pretreatment

[0062] The lyophilized powder of the AKTA purified fraction with high ACE inhibitory activity (F3) was prepared into a 2 mg / mL solution with mass spectrometry grade water. The sample was filtered through a 0.22 μm PES (polyethersulfone) aqueous filter membrane to remove any possible small particles and protect the subsequent chromatographic column and semi-preparative liquid chromatography system.

[0063] 2) Chromatographic column

[0064] Preparative reversed-phase C18 column (YMC-Pack ODS-AQ C18, 10.0 × 250 mm, 5 µm, 12 nm);

[0065] 3) Mobile phase

[0066] Mobile phase A: Ultrapure water containing 0.1% trifluoroacetic acid;

[0067] Mobile phase B: chromatographic grade acetonitrile containing 0.1% trifluoroacetic acid;

[0068] 4) System equilibration and sample loading: Start the liquid chromatography system and set the flow rate to 2.0 mL / min; first, use the initial mobile phase (5% B phase, 95% A phase) to equilibrate the column until the detector (UV detector, 214 nm) baseline is stable; then, use the manual injection valve to inject the sample, with an injection volume of 2 mL; the elution program is 0-40 min, 5-60% B, 40-60 min, 60-5% B;

[0069] 5) Sample collection and activity determination

[0070] The collection program was set to collect by volume, with a collection volume of 1 mL per tube. After completion, the liquids in different collection tubes were mixed according to the chromatographic peaks to collect different fractions, which were then freeze-dried. The in vitro ACE inhibitory activity of the different fractions of the lyophilized peptide powder was determined by UPLC. The T2 fraction showed better ACE inhibitory activity, with a retention time of 15.642 min and a peak area of ​​30280.67 mAu*s. The amino acid sequence information was determined by LC-MS / MS.

[0071] Experimental Results: Moringa seed polypeptide samples hydrolyzed with alkaline protease / trypsin for 0.1 h were initially separated using ultrafiltration. After lyophilization, in vitro ACE inhibitory activity was determined. Results showed that fractions >30 kDa, 10-30 kDa, 3-10 kDa, 1-3 kDa, and <1 kDa obtained after ultrafiltration of the Moringa seed hydrolysate all exhibited significant ACE inhibitory effects. Considering both the ACE inhibition rate and the binding mechanism of small molecule polypeptide drugs to ACE targets, fractions <1 kDa were selected for further purification using Sephadex G-50 gel filtration chromatography. Three fractions were obtained after purification. In vitro ACE inhibitory activity experiments revealed that fraction F3 had higher activity. Further separation and purification using semi-preparative liquid chromatography (SLC) yielded different fractions, T1–T9. Figure 3 The in vitro ACE inhibitory activity of the nine components was measured, and the T2 component was found to have higher activity. Figure 4 Then, it was subjected to subsequent mass spectrometry identification.

[0072] Example 3: Sequence identification and activity evaluation of a novel Moringa seed ACE C domain repressor peptide

[0073] In this embodiment, the high ACE-inhibiting component (T2) obtained after semi-preparative liquid chromatography separation and purification was identified by LC-MS / MS. The identified Moringa peptide sequence was analyzed using computer-aided analysis methods through online databases and other professional websites to screen for peptide sequences with better activity for solid-phase synthesis. Furthermore, the purified peptide after solid-phase synthesis was screened for activity using a fluorescence-based in vitro ACE C / N domain high-throughput fluorescent substrate screening method, ultimately yielding a single peptide (MO-18) specifically targeting the ACE C domain.

[0074] (1) Computer-aided prediction and virtual screening of ACE inhibitory peptides from Moringa seeds

[0075] The peptide sequences identified by mass spectrometry were organized into a standardized FASTA or plain text format, one line per column, for easy batch processing. Computer-aided automated searches were performed in the BIOPEP-UWM database (https: / / biochemia.uwm.edu.pl / biopep / start-biopep.php), SATPdb database (http: / / crdd.osdd.net / raghava / satpdb / ), AHTPDB database (https: / / ngdc.cncb.ac.cn / databasecommons / database / id / 135), and PubMed database to exclude previously reported peptide sequences. Antihypertensive activity was predicted using the AHTPIN (https: / / webs.iiitd.edu.in / raghava / ahtpin / ) website, and SVM scores were applied; higher scores indicated greater potential for antihypertensive peptides. The Peptide Ranker (http: / / distilldeep.ucd.ie / PeptideRanker / ) website was used to predict and score the bioactivity of the peptide sequences; higher scores indicated stronger potential bioactivity. The toxicity of peptide sequences was predicted using the Toxin Pred online website (https: / / webs.iiitd.edu.in / raghava / toxinpred / ). Physicochemical properties such as theoretical isoelectric point, water solubility, hydrophobicity, and stability of the peptides were predicted using online software such as Expasy (https: / / web.expasy.org / protparam / ) and PepDraw (https: / / www.pepdraw.com / ).

[0076] (2) Activity verification of candidate Moringa seed ACE C domain repressor peptide

[0077] The activity of candidate peptides was validated using an in vitro high-throughput assay based on fluorescent substrates to determine the activity of ACE C / N domains. The entire procedure was performed on black 96-well plates using fluorescence resonance energy transfer (FRET) substrates: Abz-LFK(Dnp)-OH for the ACE C domain and Abz-SDK(Dnp)P-OH for the N domain. The buffer was a 0.1 M Tris-HCl buffer (pH 7.0) containing 50 mM NaCl and 10 μM ZnCl2. The specific procedure was as follows: 40 μL of peptide was pre-incubated with 60 μL of 9 μM FRET substrate at 37°C for 10 min, followed by the addition of 20 μL of rabbit-derived ACE enzyme with an activity unit of 0.03 U to initiate the reaction. The activity was measured using a high-sensitivity multimode plate reader at 37°C. ex / λ em Continuous detection at 320 / 420 nm for 30 min was performed, and the increase in fluorescence caused by FRET substrate cleavage was recorded. Hydrolysis was considered complete when the fluorescence intensity reached a constant value. A control group without peptide was set up as a control sample with 100% enzyme activity. Captopril was set up as a positive control group. A blank sample without enzyme was set up to represent non-enzymatic degradation of the substrate (this fluorescence value should be subtracted from all treatment group samples).

[0078] Experimental Results: The Moringa seed polypeptide sequences identified by mass spectrometry were subjected to computer-aided activity prediction and analysis using online software. This included prediction of AHT hypertension activity, SVM scoring, bioactivity scoring, and prediction of parameters such as hydrophobicity, toxicity, and hydrophilicity. Based on the amino acid sequence composition patterns of highly active ACE inhibitory peptides reported in the literature, nine polypeptides—VTRLTRP, LAATHGLYVY, FFKRLLRDSK, LSSSLLCTTKLP, IAHHHLAIAILF, PLVRRAIY, PGRQPAFQ, PVMLVHFP, and SMNCMTPF—were initially selected for solid-phase synthesis for further screening. As shown in Table 2, at a polypeptide concentration of 10 μM, all nine novel candidate Moringa seed polypeptides exhibited significant ACE inhibitory effects. Among them, PLVRRAIY showed the highest ACE C domain inhibitory activity, at 77.4% (…). Figure 5 The inhibitory activity of the ACE N domain was 36.1%. Figure 6 This indicates that PLVRRAIY has selective inhibitory activity against the C-domain of ACE. Analysis of the PLVRRAIY peptide sequence revealed that the C-terminal amino acid is tyrosine and the N-terminal amino acid is proline, consistent with the amino acid composition pattern of highly active ACE inhibitory peptides. Furthermore, computer-aided activity prediction results suggest that PLVRRAIY has a potential antihypertensive effect.

[0079] Table 2. ACE inhibitory activity and physicochemical properties of candidate Moringa seed antihypertensive peptides

[0080]

[0081] Example 4: Sequence modification and in vitro digestion of a novel Moringa seed ACE C domain repressor peptide

[0082] Due to the unique amino acid composition of the MO-18 polypeptide, it is easily degraded by proteases in the gastrointestinal tract. Therefore, in this embodiment, the amino acid sequence of the MO-18 polypeptide is designed and modified to improve its stability in the gastrointestinal tract without significantly affecting its ACE inhibitory activity, which will help to facilitate the widespread application of Moringa seed polypeptide.

[0083] Based on the MO-18 peptide, the peptide sequence was modified by replacing amino acid sites easily cleaved by pepsin, trypsin, and α-chymotrypsin in the gastrointestinal tract with other hydrophobic amino acids that are not easily cleaved, thereby enhancing the stability of the peptide. In vitro digestion experiments of pepsin, trypsin, and α-chymotrypsin were conducted on the unmodified MO-18 and the modified MO-181, MO-182, MO-183, and MO-186. The experimental system was 1 mL, and the buffer was 1× phosphate buffer (pH 7.2-7.4). The volume required to digest 300 μg (100 mM) of peptide was calculated. After adjusting the solution to the optimal pH for each enzyme, the three proteases were added at an enzyme / substrate ratio of 1:20 (w / w) (3 biological replicates). A blank control group without protease was set up. Enzymatic digestion was carried out at 37℃ for 2 h, followed by inactivation at 100℃ for 10 min and freeze-drying. The lyophilized powder was reconstituted with 1 mL of methanol, centrifuged at 12000 rpm for 15 min at 4 °C, and the supernatant was collected and filtered through a 0.22 μm filter membrane. The sample was then analyzed using a TripleTOF™ 5600 system from ABSCIEX, USA, with an injection volume of 10 μL.

[0084] The formula for calculating the in vitro enzymatic hydrolysis rate is as follows:

[0085]

[0086] In the formula: R: in vitro enzymatic hydrolysis rate of the polypeptide (%)

[0087] A: Peak area (Area (counts)) of characteristic peptide ions in the blank control group

[0088] B: Area (counts) of characteristic peaks of polypeptide ions in the enzymatic digestion group.

[0089] Experimental Results: The amino acid sequence of the PLVRAIY (MO-18) peptide was modified and optimized, and its in vitro ACE inhibitory activity and simulated gastrointestinal degradation were tested. Table 3 shows that at a concentration of 10 μM, the in vitro ACE inhibitory activity of the modified PIVNNAIY (MO-181), PVVNNAIY (MO-182), PPVNNAIY (MO-183), and PPVQQAIY (MO-186) remained at approximately 50%, while their degradation rates by the three gastrointestinal proteases were significantly improved. Considering their solubility and other physical properties, MO-18 and MO-186 peptides were selected for acute animal experiments.

[0090] Table 3. ACE inhibitory activity and in vitro proteolytic rate of PLVRRAIY and its modified peptides

[0091]

[0092] Example 5: Acute animal experiment on a novel Moringa seed ACE C domain repressor peptide

[0093] This example illustrates the effect of Moringa seed ACE inhibitory peptide on spontaneously hypertensive rats. All experimental animals were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0094] Methods: Sixteen male spontaneously hypertensive rats (SHR), aged 12 weeks and weighing 250 ± 20 g, and eight male Wistar-Kyoto rats (WKY), aged 12 weeks and weighing 250 ± 20 g, were used. Before the experiment, two rats were randomly assigned to each cage. Light and darkness were controlled for 12 hours each day at a temperature of 24°C. Rats were fed a standard irradiated sterilized diet for one week to allow for acclimatization, during which blood pressure was measured to minimize stress during the experiment. Before each measurement, the rats were placed in empty cages and preheated on a heating plate for 10 minutes. Then, CODA from Kent Scientific (Shanghai Zander Medical Instruments Co., Ltd.) was used. TM The non-invasive high-channel blood pressure system was used for measurement, with each measurement set to 15 cycles, and at least 5 sets of valid data were collected from each rat.

[0095] SHR rats were randomly divided into two groups of four each. Oral administration was used to minimize harm to the rats. The three groups were: a model group receiving sterile saline, a peptide group receiving Moringa seed polypeptide intervention, and a WKY rat control group receiving saline. Specific groupings are as follows:

[0096] Control group: WKY, 1 mL sterile saline;

[0097] Model group: SHR, 1 mL sterile saline;

[0098] Peptide group: SHR, 1 mL MO-18 or MO-186 (40 mg / kg·bw).

[0099] Single-dose experiment: The rats were administered the drug by gavage according to the above-designed dose, and their blood pressure was continuously monitored at time points of 2 h, 4 h, 6 h, 8 h, 12 h, 14 h and 24 h after administration.

[0100] Experimental Results: Blood pressure changes in rats after a single dose of MO-18 polypeptide were as follows: Figure 9 As shown in the figure, systolic blood pressure dropped to a minimum of 174.54 mmHg 10 h after administration of MO-18 peptide, a decrease of 26.37 mmHg compared to pre-administration blood pressure. The blood pressure changes in rats after a single dose of MO-186 peptide are shown in the figure. Figure 10 As shown, systolic blood pressure dropped to a minimum of 182.73 mmHg 8 hours after administration of peptide MO-186, and remained at this level for 2 hours before gradually returning to normal. Compared to pre-administration blood pressure, it decreased by 15.66 mmHg. These results indicate that the novel natural antihypertensive peptides PLVRAIY (MO-18) and PPVQQAIY (MO-186) from Moringa seeds both have significant antihypertensive effects.

[0101] In summary, this invention establishes a process system for preparing ACE inhibitory peptides from crude moringa seed protein. The crude moringa seed protein was treated with an alkaline protease / trypsin combined enzymatic hydrolysis method, and the resulting peptide mixture exhibited the best in vitro ACE inhibitory activity. Results showed that small molecule peptides with a molecular weight less than 1 kDa had a more significant potential antihypertensive effect. Through structural identification, activity screening, and solid-phase synthesis, nine novel moringa seed ACE inhibitory peptides with antihypertensive potential were finally obtained. Comparative analysis revealed that peptide PLVRRAIY exhibited the strongest in vitro specific inhibitory activity against the ACE C domain, with an inhibition rate of 77.4%. To further enhance its resistance to trypsin hydrolysis, the amino acid sequence of this peptide was modified to obtain the modified peptide PPVQQAIY. Molecular and animal experiments both showed that the original peptide PLVRRAIY and the modified peptide PPVQQAIY had significant ACE inhibitory activity and antihypertensive effects, and the modified peptide showed enhanced resistance to protease degradation in the gastrointestinal environment and significantly improved stability. This invention identifies and optimizes two novel bioactive peptides from crude protein in Moringa seeds, providing new ideas for the development of natural antihypertensive polypeptide drugs and demonstrating significant application potential and research value.

Claims

1. A Moringa seed polypeptide with specific inhibitory activity against angiotensin-converting enzyme, characterized in that, The polypeptide is PLVRRAIY.

2. The use of the Moringa seed polypeptide according to claim 1 in the preparation of drugs for the prevention or treatment of hypertension.

3. A modified moringa seed natural polypeptide resistant to trypsin hydrolysis, characterized in that, The modified peptide is PPVQQAIY.

4. The use of the Moringa seed natural polypeptide modified peptide according to claim 3 in the preparation of drugs for the prevention or treatment of hypertension.

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