Broccoli antihypertensive peptide with protection effect on heart and cerebral vessels as well as preparation method and application thereof

Abstract

The invention discloses broccoli antihypertensive peptide with a protection effect on heart and cerebral vessels as well as a preparation method and application thereof. The broccoli antihypertensive peptide comprises a peptide fragment HLVNK, a peptide fragment LEHGF, a peptide fragment LLDGR, a peptide fragment HLEVR and a peptide fragment LTEVR. According to the invention, the influence of ACEI peptide on AngII level of VSMCs and the influence of proliferation, migration and apoptosis of VSMCs are explored through cell experiments. The novel broccoli ACEI peptide capable of playing a role in protecting heart and cerebral vessels in a high-glucose model is screened and identified, a foundation is laid for practical application of the novel broccoli ACEI peptide, and a good reference is provided for low-toxicity safe and effective antihypertensive peptide in the aspect of protecting the heart and cerebral vessels.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and mainly relates to broccoli antihypertensive peptides having cardiovascular and cerebrovascular protective effects, and a preparation method and application thereof, including the screening and identification of antihypertensive peptides and the research on the antihypertensive effects and cardiovascular and cerebrovascular protective mechanisms. Background Art

[0002] Broccoli is a globally important crop recognized for its health benefits due to its multiple bioactive compounds and high protein content. Bioactive peptides extracted from broccoli protein show potential to alleviate cardiovascular and metabolic diseases. ACE is a key enzyme in the human renin-angiotensin system (RAS) that regulates blood pressure. By inhibiting ACE activity, it can reduce the production of angiotensin II (Ang II), thereby inhibiting vasoconstriction caused by the binding of Ang II to receptors on the vascular wall. Under high glucose conditions, the increase in ACE activity promotes inflammation and oxidative stress, mainly by upregulating angiotensin II type 1 (AT1) receptors, accelerating the production of Ang II in atherosclerosis. Increasing evidence shows that the proliferation and migration of vascular smooth muscle cells (VSMCs) are regulated by angiotensin II (Ang II) produced by ACE.

[0003] Previous studies have shown that broccoli is the vegetable with the highest angiotensin I converting enzyme (ACE) inhibitory activity among 50 vegetables. Some ACE inhibitory (ACEI) peptides extracted from broccoli protein hydrolysate can significantly reduce blood pressure in spontaneously hypertensive rats and glucose levels in mouse models. This finding suggests that ACEI peptides in broccoli have the potential to improve hypertension and hyperglycemia.

[0004] In the context of high glucose, various strategies have been used to improve and regulate the effects of ACE and Ang II. Angiotensin II type 2 receptor signaling generally promotes vasodilation or growth inhibition, relieving vasodilation and inflammation. ACE can also protect human islets from significant glucose toxicity, which are effective strategies to lower blood glucose levels. However, each method has potential side effects, such as cough, decreased renal function, increased blood potassium, gastrointestinal irritation, and increased cardiovascular risk. At the same time, safe and effective natural bioactive peptides have attracted increasing attention, among which natural ACEI peptides can help alleviate diabetes and its related complications. Therefore, the development of natural ACEI peptides with low side effects is an urgent need for cardiovascular treatment. Summary of the invention

[0005] Based on the above problems, the present invention simulates the absorption characteristics of polypeptides using in vitro gastrointestinal simulation and Caco-2 cell transport model. Using a method combining peptidomics and molecular docking, ACEI peptides with high absorption potential and antihypertensive activity were screened from broccoli. Finally, vascular smooth muscle cells (VSMCs) under high sugar conditions were further studied. In view of this, the present invention proposes a broccoli antihypertensive peptide with a protective effect on cardiovascular and cerebrovascular diseases, as well as a preparation method and application thereof.

[0006] In view of this, the present invention provides broccoli antihypertensive peptide having cardiovascular and cerebrovascular protective effects, and a preparation method and application thereof.

[0007] In order to achieve the above purpose, the present invention adopts the following technical solutions:

[0008] The broccoli antihypertensive peptides that have a protective effect on cardiovascular and cerebrovascular diseases are the following peptides:

[0009] The amino acid sequence of the first peptide segment is HLVNK;

[0010] The amino acid sequence of the second peptide segment is LEHGF;

[0011] The amino acid sequence of the third peptide segment is LLDGR;

[0012] The amino acid sequence of the fourth peptide segment is HLEVR;

[0013] The amino acid sequence of the fifth peptide segment is LTEVR.

[0014] The broccoli antihypertensive peptides include peptide HLVNK, peptide LEHGF, peptide LLDGR, peptide HLEVR and peptide LTEVR, five ACE inhibitory peptides, with high ACE inhibitory activity and digestion stability greater than 90%. The application of the five ACE inhibitory peptides in cardiovascular and cerebrovascular protection can play a cardiovascular protective role under a hyperglycemia model.

[0015] The method for preparing the broccoli antihypertensive peptide having a protective effect on cardiovascular and cerebrovascular diseases adopts proteomics and molecular docking for preparation and screening, and uses a hyperglycemia model to study the protective effect on cardiovascular and cerebrovascular diseases, comprising the following steps:

[0016] (1) Preparation of broccoli protein and simulated in vitro digestion;

[0017] (2) The digestate in step (1) is subjected to an ACE inhibitory activity assay, the digestate in step (1) is subjected to a preliminary screening using mass spectrometry, and then molecular docking is used to perform a final screening to identify peptides with potential ACE inhibitory activity, i.e., broccoli antihypertensive peptides with cardiovascular and cerebrovascular protective effects.

[0018] In step (1), broccoli protein preparation and in vitro simulated digestion specifically include:

[0019] The fresh broccoli stems and leaves are juiced, the juice is centrifuged, the precipitate is centrifuged and collected, and freeze-dried to obtain a broccoli protein sample. Subsequently, the hydrolyzate after hydrolysis with papain is simulated digested and the supernatant is collected, and the supernatant is the digestate.

[0020] In step (1), the centrifugal separation conditions are: centrifugal separation at 4500-5500 rpm for 5-15 min at 2-6° C. The centrifugal collection conditions are: centrifugal collection at 7500-8500 rpm for 5-15 min at 2-6° C.

[0021] The application of the broccoli antihypertensive peptide having cardiovascular and cerebrovascular protective effects in the preparation of cardiovascular and cerebrovascular protective drugs or health products.

[0022] The present invention provides an application of the broccoli ACEI peptide as described above in protecting cardiovascular and cerebrovascular vessels.

[0023] Through the above technical scheme, it can be seen that the beneficial effects achieved by the present invention are: the present invention explores the effects of ACEI peptides on AngII levels of VSMCs and the effects on proliferation, migration and apoptosis of VSMCs through cell experiments. The present invention not only screens and identifies new broccoli ACEI peptides that can play a cardiovascular and cerebrovascular protective role in a high-sugar model, laying a foundation for its practical application, but also provides a good reference for low-toxic, safe and effective antihypertensive peptides in protecting cardiovascular and cerebrovascular. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0025] Figure 1 The attached figure shows the expected interaction between the peptide and the ACE molecule, wherein A is HLEVR, B is HLVNK, C is LLDGR, D is LTEVR, and E is LEHGF;

[0026] Figure 2 The attached figure shows the effect of ACEI peptide on Ang II level in vascular endothelial cells cultured under high glucose conditions;

[0027] Figure 3 The attached figure shows the effects of ACEI peptide on VSMC proliferation, migration and apoptosis induction under high glucose conditions;

[0028] Figure 4The attached figure shows the effect of ACEI peptide on ERK and p38 activation under high glucose conditions. DETAILED DESCRIPTION

[0029] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0030] The drugs required for the present invention are conventional experimental drugs, which are purchased from commercial channels; the experimental methods not mentioned are conventional experimental methods and will not be described in detail here.

[0031] A method for preparing broccoli antihypertensive peptide having a protective effect on cardiovascular and cerebrovascular diseases comprises the following steps:

[0032] (1) Preparation and in vitro digestion of broccoli protein:

[0033] Fresh broccoli stems and leaves were juiced and centrifuged at 5000 rpm for 10 min at 4°C. The precipitate was collected by centrifugation at 8000 rpm for 10 min at 4°C and freeze-dried to obtain a broccoli protein sample. Subsequently, the hydrolyzate after hydrolysis with papain was simulated digested and the supernatant was collected.

[0034] (2) Determination of ACEI activity of broccoli peptides before and after digestion;

[0035] (3) Simulated absorption of peptides in broccoli hydrolysate:

[0036] The Caco-2 cell model was used to simulate the absorption of peptides in broccoli enzymatic hydrolysate, and the transepithelial electrical resistance (TEER) on the monolayer was measured every two days by a cell resistance meter. The culture medium containing the broccoli enzymatic hydrolysate suspension on the AP side and the basolateral side was collected for subsequent analysis;

[0037] (5) Screening and synthesis of ACEI peptides from broccoli hydrolysate:

[0038] The (1) hydrolysates were initially screened using mass spectrometry, and then molecular docking was used for the final screening to identify potential ACEI peptides (HLVNK, LEHGF, LLDGR, HLEVR and LTEVR).

[0039] (6) Culture of VSMCs:

[0040] The thoracic aorta of male Sprague-Dawley rats was dispersed in a HBSS solution containing 1 mg / mL collagenase II and 0.5 mg / mL elastase I. The mixture was digested for 90 min to obtain suitable VSMCs. The obtained VSMCs were then cultured in a humidified atmosphere of 37°C, 5% CO2 in DMEM supplemented with 10% FBS, 2 mmol / L glutamine and 1 g / L d-glucose. The purity of VSMCs was evaluated using the smooth muscle cell-specific marker SM22α. All VSMCs used in the experiment were between the 3rd and 8th generations.

[0041] (7) CCK-8 assay;

[0042] (8) EdU detection of VSMCs:

[0043] The cells were seeded in a 12-well plate, cultured overnight, serum deprived for 24 hours, and then 400 μL of culture medium was taken and 200 μL of 2X EdU solution was added to each well and incubated at room temperature for 2 hours. The cells were fixed with 4% paraformaldehyde for 15 minutes and then permeabilized with 0.3% Triton X-100 in PBS for 15 minutes. The cells were then washed with 3% BSA in PBS. 200 microliters of 1X click reaction mixture was added to each well and the cells were washed again. The nuclei were incubated with 1X Hoechst 33342 and the cells were washed again. Finally, the cells were photographed at 50 times magnification using a fluorescence microscope;

[0044] (9) VSMCs migration assay:

[0045] VSMCs were seeded in 6-well plates and cultured to 80% confluence, then serum deprived for 24 h. After serum starvation, a standard wound (<3 mm) was formed using a 200 μL sterile plastic pipette tip, and fresh culture medium was replaced three times. VSMCs were cultured in DMEM with 2% fetal bovine serum, and wound images were taken at 0 h and 24 h.

[0046] (10) Apoptosis analysis of VSMCs:

[0047] Cell apoptosis was detected using the FITC-Annexin V apoptosis detection kit;

[0048] (11) Western Blot Analysis:

[0049] Proteins were extracted with RIPA buffer. Equal amounts of protein were separated by 8-10% SDS-PAGE and then transferred to 0.22 μm PVDF membranes. The membranes were blocked with 5% skim milk and incubated overnight with appropriate primary antibodies. Enzyme-labeled secondary antibodies were then applied for binding and visualization by Western blot luminol reagent. Densitometry quantification was performed using ImageJ-V1.8.0.

[0050] Example 1

[0051] (1) Mass spectrometry

[0052] Proteomics and peptidomics improve the accuracy of the extraction and purification process through comprehensive analysis of protein composition and content, thus providing a theoretical basis for the development of functional ingredients and promoting the development of new health products. Since the cleavage effect of pepsin is weak and the cleavage effect of pancreatic enzymes is strong, the c-terminal amino acids of the obtained peptide sequences are almost all arginine (Arg) and lysine (Lys). A high ratio of Arg to Lys can reduce the systolic blood pressure of hypertensive rats. Two protein sequences rich in bioactive peptides were selected for subsequent peptide identification. The mass spectrometry identification results of broccoli peptides are shown in Table 1.

[0053] Table 1. Mass spectrometry analysis results of broccoli peptides

[0054]

[0055] (2) Molecular docking

[0056] To investigate the molecular interaction between broccoli ACEI peptide and ACE, we performed a visual analysis of the molecular docking results ( Figure 1 ). The interaction between broccoli peptides and ACE is mainly mediated by hydrogen bonding interactions, hydrophobic interactions and electrostatic interactions. Among them, hydrogen bonding plays a key role in the formation of the complex between ACE pentapeptide and amino acid residues. Specifically, 11, 12, 10, 10 and 8 hydrogen bond peptides (HLVNK, LEHGF, LLDGR, HLEVR and LTEVR) were generated between ACE and ACE. Amino acid residues such as GLU384, TYR523, ASP415, LYS511, GLN281, ASN277, THR282, ASP453 are positioned in the ACE active site through hydrogen bonds. In addition, HLEVR, LTEVR, LLDGR and HLVNK interact with Zn in ACE. 2+ The prediction results of ACEI peptides are shown in Table 2. The results showed that these five peptides have potential ACEI activity.

[0057] Table 2. ACE binding energy of broccoli peptides

[0058]

[0059] (3) Determination of ACE inhibitory activity and digestive stability of peptides

[0060] The ACEI activity and digestion stability of the peptides are shown in Table 3. The results showed that all five peptides had ACEI activity and digestion resistance.

[0061] Table 3. ACEI activity and digestive stability of peptides

[0062]

[0063] Example 2

[0064] (1) Effect of Ang II levels in VSMCs

[0065] Figure 2 The effects of Ang II on VSMCs under high glucose conditions are shown. Figure 2 AG shows the effect of AngII treatment on the expression of related proteins in VSMCs under high glucose (HG) conditions. Ang II significantly increased the expression of PCNA and MMP2, indicating enhanced cell proliferation and matrix remodeling. In addition, Ang II upregulated the pro-apoptotic protein Bax and downregulated the anti-apoptotic protein Bcl2, indicating that Ang II treatment promoted cell apoptosis. Figure 2 F shows that under HG conditions, the Ang II protein level was significantly higher than that under normal glucose (NG) conditions, and the increase was particularly significant at 24 h, indicating that high glucose induced an increase in Ang II expression. Figure 2 G shows that AngII levels did not change but changed significantly at different time points under HG conditions.

[0066] Figure 2 HM showed the effects of different peptides on cell viability and Ang II levels under HG conditions. The results showed that HLLVNK, LEHGF, HLEVR, and LLDGR at a concentration of 0.5 mg / mL could significantly increase cell viability, indicating that these peptides have a protective effect on high glucose-induced cell damage and can promote cell proliferation. In contrast, the protective effect of LTEVR was not significant. Figure 2 M highlighted the effects of peptide combinations on Ang II levels under HG conditions, showing that various combinations (such as HG+HLLVNK, HG+LTEVR) significantly reduced Ang II levels. This suggests that these peptides may have a potential protective role in alleviating high glucose-induced cellular stress and inflammation.

[0067] Collectively, these peptides not only promote cell proliferation but also effectively reduce cellular stress by reducing Ang II levels. Compared with the general population, patients with diabetes have higher cardiovascular morbidity and mortality. In the present study, Ang II levels and markers associated with phenotypic switching (such as PCNA, MMP2, Bcl2, SM22α, and BAX) in VSMCs were significantly altered under high glucose conditions, suggesting glucose-induced dysfunction. The ACEI peptides HLVNK, LTEVR, LEHGF, LLDGR, and HLEVR were found to effectively reduce VSMCs viability and had a significant inhibitory effect on intracellular Ang II levels. These findings suggest that these ACEI peptides may counteract the adverse vascular effects of elevated glucose by downregulating Ang II within VSMCs, highlighting their therapeutic potential in controlling glucose-induced vascular complications.

[0068] (2) Effects of peptides on proliferation, migration and apoptosis of VSMCs

[0069] Effects of ACEI peptides on proliferation, migration and apoptosis of vascular smooth muscle cells Figure 3 shown. Figure 3 AE are Western blot results of PCNA, MMP2, Bcl2, and Bax protein expression under different conditions. In the HG group, PCNA and MMP2 expression were significantly decreased, Bcl2 expression was decreased, and Bax expression was increased. In contrast, treatment of HLVNK, LTEVR, LEHGF, LLDGR, and HLEVR with peptides significantly restored the expression of these proteins, indicating that the peptides have a protective effect on high glucose-induced cell damage. Figure 3 F shows the cell proliferation assessed by BrdU and Hoechst staining. In the peptide-treated groups, especially HLLVNK and LEHGF, the BrdU-positive cells increased significantly, indicating enhanced proliferation. Figure 3 G further quantified the BrdU-positive cells, which further supported that peptide treatment significantly promoted cell proliferation. Figure 3 H shows the results of the scratch assay, where peptide treatment significantly improved cell migration at 24 h compared with the HG group, indicating that the peptide enhanced cell migration. Figure 3 I quantified the rate of wound closure, showing that peptide treatment significantly accelerated wound healing. Figure 3 J and 3K evaluated apoptosis by flow cytometry, showing that apoptosis was significantly increased under HG conditions and significantly reduced by peptide treatment, confirming their protective role in alleviating high glucose-induced apoptosis. EdU and migration experiments showed that these peptides effectively reduced the proliferation and migration of VSMCs, and flow cytometry showed that these peptides promoted apoptosis.

[0070] The inhibition of proliferation and migration, coupled with the induction of apoptosis, suggests that these peptides have broad regulatory effects that may not be limited to ACE inhibition. Studies have shown that this effect may be mediated through the MAPK / ERK pathway, where reduced phosphorylation was observed, disrupting the hyperglycemia-driven signaling that normally drives VSMC activity and vascular remodeling. In addition, ACEI peptides modulate the expression of Ang II and ACE2, potentially enhancing their cardioprotective effects by lowering blood pressure and attenuating Ang II-mediated inflammatory and proliferative signals.

[0071] (3) Effects of ACEI peptides on ERK and p38 activation in VSMCs

[0072] Effects of ACEI peptides on ERK and p38 activation in vascular smooth muscle cells Figure 4 Western blot results showed that Ang II treatment significantly changed the expression levels of ACE, AT1R, ACE2, p38, p-p38 and ERK1 / 2 ( Figure 4 A), indicating that Ang II through these signaling molecules and pathways. Specifically, quantitative analysis showed that Ang II significantly up-regulated ACE ( Figure 4 B) and AT1R ( Figure 4 C), which means that Ang II exerts its biological effects by enhancing the expression of these key proteins. In addition, ACE2 expression was significantly reduced after Ang II treatment ( Figure 4 D), indicating that AngII may aggravate metabolic disruption or cell damage by negatively regulating ACE2. p38 signaling pathway is activated ( Figure 4 E), p-p38 expression increased, emphasizing the importance of this pathway in Ang II-induced stress response. ERK1 / 2 pathway was also affected ( Figure 4 F), Increased expression of p-ERK1 / 2 indicated that it promoted stress response through Ang II-induced signaling pathway. Figure 4 G shows the changes in protein expression under high glucose and peptide treatment conditions, illustrating the regulatory effects on ACE, AT1R, ACE2, and p38. Notably, peptide treatment, especially HGLVNK and HGLTEVR, modulated the expression of ACE ( Figure 4 H), indicating that these peptides partially counteracted Ang II-induced upregulation of ACE. Changes in AT1R expression in the peptide-treated groups ( Figure 4 I) suggests that the peptide may regulate AT1R levels to mitigate the effects of Ang II. In the peptide-treated group, ACE2 expression was restored ( Figure 4 J), indicating that the peptide can counteract the negative effects of Ang II by enhancing ACE2 expression. In addition, p-p38 expression was decreased in the peptide-treated group ( Figure 4K) showed that these peptides inhibited excessive p38 pathway activation, thereby alleviating cellular stress responses. Modulation of the p-p38 pathway further indicated that ERK1 / 2 activation in the peptide-treated group ( Figure 4 L), reflected by decreased expression of p-ERK1 / 2.

Claims

1. A broccoli antihypertensive peptide having a protective effect on cardiovascular and cerebrovascular diseases, characterized in that: The following peptides: The amino acid sequence of the first peptide segment is HLVNK; The amino acid sequence of the second peptide segment is LEHGF; The amino acid sequence of the third peptide segment is LLDGR; The amino acid sequence of the fourth peptide segment is HLEVR; The amino acid sequence of the fifth peptide segment is LTEVR.

2. The method for preparing the broccoli antihypertensive peptide having cardiovascular and cerebrovascular protective effects according to claim 1, characterized in that: The following steps are involved: Proteomics and molecular docking were used for preparation and screening, and the hyperglycemia model was used to study the cardiovascular and cerebrovascular protective effects. The method was carried out in the following steps: (1) Preparation of broccoli protein and simulated in vitro digestion; (2) The digested product in step (1) is initially screened using mass spectrometry analysis, and then molecular docking is used for final screening to identify peptides with potential ACE inhibitory activity, namely, broccoli antihypertensive peptides with cardiovascular and cerebrovascular protective effects.

3. The method for preparing the broccoli antihypertensive peptide having cardiovascular and cerebrovascular protective effects according to claim 2, characterized in that: In step (1), broccoli protein preparation and in vitro simulated digestion specifically include: The fresh broccoli stems and leaves are juiced, the juice is centrifuged, the precipitate is centrifuged and collected, and freeze-dried to obtain a broccoli protein sample. Subsequently, the hydrolyzate after hydrolysis with papain is simulated digested and the supernatant is collected.

4. The method for preparing the broccoli antihypertensive peptide having cardiovascular and cerebrovascular protective effects according to claim 3, characterized in that: In step (1), the centrifugal separation conditions are: at 2 to 6° C., 4500 to 5500 rpm for 5 to 15 min.

5. The method for preparing the broccoli antihypertensive peptide having cardiovascular and cerebrovascular protective effects according to claim 3, characterized in that: In step (1), the conditions for centrifugal collection are: centrifugation at 7500-8500 rpm for 5-15 min at 2-6°C.

6. Use of the broccoli antihypertensive peptide having cardiovascular and cerebrovascular protective effects according to claim 1 in the preparation of cardiovascular and cerebrovascular protective drugs or health products.

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